1. Field of the Invention
The present invention relates to an amphiphilic polymer.
2. Discussion of Related Art
In proteins of a human body, a membrane protein which is present in a cell membrane accounts for 30% or more of the total genome. Particularly, a receptor such as a G-protein coupled receptor (GPCR) or an ion channel protein is responsible for a signal pathway in the living body, is involved in various diseases such as cancer, nervous system disorders, immune diseases, inflammatory diseases or the like, accounts for 60% or more of drug targeting, and occupies an important position in disease diagnosis and new drug development. The function of the above-described membrane protein is induced by an interaction with other molecules such as a ligand. Accordingly, in order to analyze the interaction between the membrane protein and the ligand or the like, a technique of preparing the refined membrane protein having an active structure and a technique of fixing the membrane protein to a support are core technologies to analyze binding specificity between the membrane protein and ligand, and are essential for the applications in biotechnology.
Although a large number of techniques have been developed for the stabilization and fixation of proteins, techniques for the refinement as well as the stabilization and fixation of the membrane protein are highly limited due to structural characteristics of the membrane protein. In the case of a water soluble protein, hydrophilic amino acid residues are mainly exposed to a surface of proteins, interact with water molecules, and thus may be easily stabilized. On the other hand, the membrane protein has many hydrophobic surfaces formed of hydrophobic residues, and thus forms a stable structure when the membrane protein is present at a lipid bilayer, positioned adjacent to hydrophobic sites of the lipid. However, when the membrane protein is present in an aqueous solution without the lipid, the hydrophobic surfaces of the membrane protein aggregate to each other without interacting with the water molecules, and thereby forming insoluble precipitates which are inactive. Accordingly, detergents which are water-soluble and are capable of stabilizing the hydrophobic surfaces of the membrane protein are required in the refinement process for research on the function of the membrane protein.
Suitable detergents are bound to the hydrophobic surfaces of the membrane protein while the active structure of the membrane protein is maintained, and thereby allow the membrane protein to maintain the active structure in the aqueous solution without aggregating to each other. However, in many types of the commercially available detergents, the number of the detergents allowing the specific membrane protein to be active in the aqueous solution is small, and a process of determining the above-described detergents is complicated. For example, few types of detergents are capable of allowing receptor proteins such as a GPCR to maintain the active structure in the aqueous solution, and although b-octylglucoside (OG) and dodecylmaltoside (DDM) are known to have a great effect in stabilizing the membrane protein under circumstances of the aqueous solution (Journal of Biological Chemistry, 2001, vol 276, pp 32403-32406. Protein Expression and Purification 2012, vol. 86 pp 12-20), the above-described detergents may not be widely applied to all membrane proteins, and a high cost is required therefor.
In addition to the detergents, methods of forming a bicelle (Analytical Biochemistry, 2000, vol. pp284, 327-333) and a nanodisc (Nano Letter 2002, vol 2, pp 853-856) using lipids and detergents or lipids and lipid-stabilizing proteins, respectively, are used as a method of stabilizing the membrane protein in the aqueous solution. According to these methods, the membrane protein forms a structure having a size of several nanometers in a similar environment to the lipid bilayer, the membrane proteins may be maintained in a state of the aqueous solution under the lipid layer environment. However, a manufacturing process of the above-described method is complicated, and a high coat is required therefor.
A method of stabilizing the membrane protein using a polymer has also been known. The polymer referred to as an amphipol is a polymer which has both hydrophobic sites and hydrophilic sites in one polymer molecule by introducing an alkyl group into a polyacrylic acid, and has been reported to have a high effect in the membrane protein stabilization (Biochemistry, 2006, vol. 45, pp 13954-13961). However, the above-described polymer has a low production yield, and lipid layer-reassembling activity of the membrane protein has not yet been reported. Further, the manufacturing cost is high due to a low synthesis yield of the amphipol, and the lipid layer-reassembling function of the membrane protein with the amphipol has not yet been reported. Further, when the amphipol is used, a process of introducing a functional group such as a fluorescent material, biotin, or the like which may be used in the process of property analysis and fixation of the proteins to a specific site of the polymer is complicated and difficult.
Accordingly, there is a need for techniques of stabilizing the membrane protein in the aqueous solution which may be widely applied to the membrane proteins such as receptor proteins and ion channel proteins which serve an important role in the living body and are target proteins in new drug development. The embodiment of the present invention is intended to develop a method of preparing a polymer which is an amphiphilic polymer having both hydrophilic and hydrophobic sites, and includes a functional group for fixation at the same time by introducing the functional group for fixation, a hydrophobic functional group, and a hydrophilic functional group into a poly-glutamic acid, and is intended to develop techniques of the membrane protein stabilization, the membrane protein fixation, and lipid layer-reassembling of the membrane protein.